Amelioration of Murine Colitis by Attenuated Salmonella choleraesuis Encoding Interleukin-19

The imbalance of mucosal immunity in the lower gastrointestinal tract can lead to chronic inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis. IBD is a chronic inflammatory disorder that causes small and/or large intestines ulceration. According to previous studies, recombinant interleukin (IL)-10 protein and genetically modified bacteria secreting IL-10 ameliorate dextran sulfate sodium (DSS)-induced colitis in mice. IL-19 is a transcriptional activator of IL-10 and can alter the balance of T helper 1 (Th)1/Th2 cells in favor of Th2. In this study, we aimed to investigate whether the expression of the murine IL-19 gene carried by Salmonella choleraesuis (S. choleraesuis) could ameliorate murine IBD. Our results showed that the attenuated S. choleraesuis could carry and express the IL-19 gene-containing plasmid for IBD gene therapy by reducing the mortality and clinical signs in DSS-induced acute colitis mice as compared to the untreated ones. We also found that IL-10 expression was induced in IL-19-treated colitis mice and prevented inflammatory infiltrates and proinflammatory cytokine expression in these mice. We suggest that S. choleraesuis encoding IL-19 provides a new strategy for treating IBD in the future.


Introduction
Uncontrolled mucosal immunity in the gastrointestinal tract of humans results in chronic inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC) [1]. IBD is a chronic inflammatory disorder in the small and/or large intestines with ulceration. Genetic, environmental, and immunologic factors can contribute to the disease. However, the exact etiology of IBD remains unclear. In general, inappropriate inflammatory responses to unknown antigens presented by the normal bacterial microflora can be found in the gut of IBD patients [2].
IL-19 belongs to the IL-10 cytokine family, which influences the maturation of T cells and alters the imbalance of Th1/Th2 cells in favor of Th2 [13]. Furthermore, IL-10 is strongly induced in IL-19-stimulated peripheral blood mononuclear cells (PBMCs), indicating that IL-19 is a transcriptional activator of IL-10 [14]. Therefore, it may suggest that IL-19 gene delivery can ameliorate murine colitis.
Salmonella typhimurium (S. typhimurium), a facultative anaerobe, has been developed as a gene delivery vector. It can transfer eukaryotic expression plasmids to mammalian cells in vitro and in vivo [15]. The oral administration of attenuated S. typhimurium carrying a eukaryotic expression plasmid encoding interferon-γ gene restores the production of this cytokine in macrophages of interferon-γ-deficient mice [16]. S. typhimurium harboring the plasmids with eukaryotic expressible IL-12 or granulocyte/macrophage colony-stimulating factor (GM-CSF) genes exerts anti-tumor effects in mice [17].
We have reported that attenuated Salmonella choleraesuis (S. choleraesuis) can serve as a DNA vaccine against pseudorabies and enhance both humoral and cellular immune responses [18,19]. Therefore, we planned to exploit S. cholersesuis carrying a eukaryotic expression plasmid encoding the IL-19 gene as a new gene therapeutic strategy in a DSSinduced murine colitis model. Through the induction of IL-10, S. cholersesuis carrying the IL-19 gene may have therapeutic potentials for treating murine colitis.
Female C57BL/6 mice (8 to 10 weeks old) were obtained from the Laboratory Animal Center of National Cheng Kung University. The animals were maintained in specific pathogen-free animal care facilities under isothermal conditions with regular photoperiods. The experimental protocol adhered to was approved by the Laboratory Animal Care and Use Committee of National Cheng Kung University (Approval numbers: 109160 and 108324).

Assays of Gene Transfer In Vitro and In Vivo
Caco-2 cells (5 × 10 5 /well) were cultured into 6-well plates for 24 h. Thereafter, S.C/pLJD-CMV-Luc and S.C. at multiplicity of infections (MOIs) of 0.1, 1, and 10 were added to cells and cultured with 1 mL of antibiotic-free medium and incubated for 8 h. After 8 h, the cells were washed, replenished with gentamycin (50 ug/mL)-containing complete medium, and cultured for 24 h. The cells were lysed to prepare extracts for the determination of the luciferase activity by a luciferase assay kit (Tropix, Bedford, MA, USA).

Induction of Colitis and Injection of S.C. in DSS-Induced Colitis Mice
Dextran sodium sulfate (DSS) [M.W. 36,000-50,000 (MP Biomedicals, LLC, Eschwege, Germany)] was added to tap water at a concentration of 2% for C57BL/6 mice. Fresh DSS solution was prepared daily. Mice were exposed to 2% DSS for six days. Healthy control mice received tap water only.
Mice receiving DSS were challenged with saline, 10 8 CFU of S.C., S.C./pLJD, or S.C./pLJD-CMV-mIL19 per mouse from day 0 to day 5 (6 doses). The schedule of the treatment is shown in Figure 1. The parameters, including survival, body weight, stool consistency, fecal bleeding, and diarrhea, were used to evaluate clinical signs in the saline, S.C.-, S.C./pLJD-, or S.C./pLJD-CMV-mIL19-treated DSS colitis mice. Figure 1. The schedule of gene therapy in DSS-induced colitis mice. The 2% DSS was added to tap water for 6 days and changed to normal tap water at day 6. At day 0, the DSS-treated mice were orally administrated with 10 8 CFU of S.C., S.C./pLJD, S.C./pLJD-CMV-mIL19 and normal saline (500 µL). The survival and body weight loss were monitored every day. The inflammatory score, diarrhea score, and fecal blood score were monitored at day 3 and day 5.

Histological Evaluation and Scoring
The colons were isolated from the mice and assessed for diarrhea and fecal blood analyses by the blind pathophysiologists. Then, the colon specimens were fixed in 10% paraformaldehyde, embedded in paraffin and sliced into sections. The sections were stained with haematoxylin and eosin (H&E), and histological analysis was performed in a blinded fashion. The pathophysiology of the tissue was characterized by the presence of ulceration, inflammatory cells (neutrophils, macrophages, lymphocytes, and plasma cells), signs of edema, crypt loss, surface epithelial cell hyperplasia, goblet cell reduction, and signs of epithelial regeneration. Diarrhea score, fecal blood score, and inflammatory score were evaluated as previously described [20].

Statistical Analysis
The data are expressed as the mean ± SEM. The differences between the two groups and among the groups were analyzed using Student's t-test and one-way ANOVA followed by Dunnet multiple comparison tests, respectively. Differences in body weight changes were compared using repeated-measures analysis of variance. The percent survival was compared using a log-rank (Mantel-Cox) test (Prism 5.0). p-values less than 0.05 were considered significant.

S.C./pLJD-CMV-mIL19 Decreased Intestinal Inflammation in DSS-Induced Acute Colitis Mice
At day 3, the control mice showed minimal changes in the surface epithelium and slight infiltration of inflammatory cells to the mucosa. At day 5, there were changes in the control mice, with a loss of crypts and a reduction in goblet cells, signs of surface epithelial regeneration, focal ulcerations, moderate infiltration of inflammatory cells to the mucosa, and edema in the submucosa. S.C./pLJD-CMV-mIL19-treated mice showed normal symptoms of mucosa but a slight infiltration of inflammatory cells to the mucosa at day 5 (Figure 6a). At day 5, the control mice showed significantly higher inflammatory scores than the S.C./pLJD-CMV-mIL19-treated mice (saline, p = 0.007, S.C., p = 0.01, S.C./pLJD, p = 0.01 in comparison to S.C./pLJD-CMV-IL19 treatment (Figure 6b). Furthermore, control mice had moderate infiltration of Mac-3-and Gr1-positive cells when compared to S.C./pLJD-CMV-mIL19 treated mice in the mucosa (Figure 7). Taken together, S.C./pLJD-CMV-IL19 treatment can reduce macrophage and neutrophil infiltrations to the mucosa in DSS-induced acute colitis mice.

Discussion
The transfer of bacteria-mediated eukaryotic expression plasmids to mammalian cells has been applied to gene therapy. Bacteria, such as S. typhimurium, S. typhi or E. coli, invade their host cells by expressing the invasion of Y. pseudotuberculosis and then remain in the vacuole. Due to metabolic reduction, they die there and release their expression plasmids. With the unknown mechanisms, the plasmids can cross the vesicular membrane and translocate into the cell nucleus of the host cells, and they are expressed there [15]. According to these findings, we showed that S. choleraesuis transferred eukaryotic expression plasmids into colon epithelial cells in vitro, resulting in transgene expression in a dose-dependent manner (Figure 2a). Furthermore, we also showed that S. choleraesuis transferred eukaryotic expression plasmids into the intestinal glands, lamina propria and muscular mucosa (Figure 2b). Thus, we designated an intracellular bacteria S. choleraesuis carrying a eukaryotic-expressing plasmid encoding IL-19 to evaluate the therapeutic effect on murine colitis. IL-19-deficient mice suffered from exacerbated experimentally induced colitis with the increased production of several proinflammatory cytokines, indicating IL-19 to be an anti-inflammatory cytokine in intestinal inflammation in mice [21,22]. To our knowledge, this should be the first report revealing murine IL-19 gene therapy via attenuated S. choleraesuis that ameliorates colitis in mice, indicating the anti-inflammatory role of IL-19 in the gastrointestinal tract. Interestingly, there were two IL-19 polymorphisms that might have protective roles in patients with UC [23]. Jordan et al. [14] demonstrated that human IL-19 regulates immunity through the auto-induction of IL-19 and the production of IL-10. We also found that the murine IL-19 gene transferred to the colon epithelial cells and the treated IL-19 conditioned medium induced IL-10 production in splenocytes and RAW264.7 ( Figure 3). However, Liao et al., showed that murine IL-19 induced the production of IL-6 and TNF-α, which contributed to apoptosis in mouse monocyte [24]. Human IL-19 has two potential N-linked glycosylation sites at positions 56 and 135 that makes multiple IL-19 protein bands around the regions of 35-40 kDa on SDS-PAGE [25]. Accordingly, it may suggest that the different glycosylated forms of IL-19 play distinct roles in immunoregulation. In this study, we showed that IL-19 was an IL-10 inducer and that IL-10 production was stimulated at the early stage of DSS-induced colitis ( Figure 4). We also found that IL-19 gene expression in the gastrointestinal tract alleviated gut inflammation through the induction of IL-10, as well as decreased the levels of proinflammatory cytokines, including TNF-α, IL-6, and IL-1β ( Figure 8).
Gut inflammation can be induced in mice using chemical compounds such as TNBS, oxazolone, and DSS. In 1990, Okayasu et al., described a model in which mice receiving DSS orally developed acute and chronic colitis resembling UC, but the underlying mechanism remained unclear [26]. Several mechanisms, including toxic effects on the epithelium, increased exposure to luminal antigens via the destruction of mucin content, and altered macrophage function due to ingestion of DSS, were proposed [27,28]. Furthermore, inflammatory infiltrates in the acute phase of DSS-induced colitis consisted predominantly of macrophages, neutrophils, and eosinophils [29]. In parallel to our findings that the S.C./pLJD-CMV-mIL19-treated DSS acute colitis mice had reduced macrophage and neutrophil infiltrations (Figure 7).
Human IBD studies provide evidence that CD is characterized by a Th1-mediated immune response [30,31]. However, Boirivant M. et al., described a murine model induced by oxazolone colitis that resembled UC, having a typical Th2-mediated response through the secretion of IL-13 by natural killer T cells [32]. It is suggested that Th1 characterized CD, whereas UC was characterized by a Th2 type immune response. In this study, our murine model of DSS-induced colitis resembling CD indicated that Th1-cytokines played important roles and were associated with excessive Th1-mediated responses [33]. IL-19 influences the maturation of T cells and alters the balance of Th1/Th2 cells in favor of Th2 [34]. It may suggest that IL-19 can alter the excessive Th1-mediated response by shifting to Th2 in DSS-induced colitis.
Prebiotics and probiotics hold promises for treating IBD, which have been proven in some studies as prescription drugs [35,36]. The oral administration of different probiotic strains, such as Lactobacillus and Bifidobacterium, prevents DSS-induced acute colitis [37]. Interestingly, our data showed that the oral administration of either S. choleraesuis or S.C./pLJD in DSS acute colitis mice can decrease TNF-α, IL-6, and IL-1β production compared with the saline group ( Figure 8). Attenuated salmonella strains can interact with model human epithelia and attenuate the synthesis of inflammatory molecules elicited by diverse proinflammatory stimuli by preventing the nuclear translocation of NF-kappaB [38]. Although we might term attenuated S. choleraesuis as a probiotic in DSS-induced colitis due to the decrease in pro-inflammation cytokines production, the modulation of the gut microbiome in DSS-induced colitis mice by S. choleraesuis could not be ruled out. Therefore, the limitation of these data is the lack of bacterial sequencing before and after each therapy. Another limitation is the lack of evidence via immunofluorescence microscopy for the presence of the GFP signals in the epithelial cells of mice. Although we prove that S.C./pLJD-CMV-mIL19 can express the IL-19 gene in the colon extracts of the colitis mice, more specific investigations to prove the in vivo colon epithelial expression of IL-19 in these mice will be required in the future.
In conclusion, the present study demonstrates for the first time that IL-19 is a gene therapy agent in a murine model of colitis. The immunoregulatory gene delivered by the attenuated salmonella strain offers the perspective of local modulation of the inflammatory responses. It may provide another choice to ameliorate IBD in the future.

Institutional Review Board Statement:
The animal study adhered to criteria approved by the Laboratory Animal Care and Use Committee of the National Cheng Kung University (Approval numbers: 109160 and 108324).

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.